This invention relates to a process for the manufacture of a flow cell for light absorption measurement, and more particularly, to an improved method for making such a flow cell whose inner wall has an index of refraction less than that of water. The flow cell has special application in the well established techniques of high performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE).
Light absorption detectors for HPLC and CZE generally comprise four basic components: a light source, a means for selecting a narrow increment of wavelengths, a flow cell, typically in the form of a hollow tube through which a sample to be analyzed and the light are passed, and a light sensor which measures the amount of light transmitted through the flow cell. When a light absorbing component passes through the flow cell, the amount of light transmitted through the flow cell decreases in accordance with Beer's law: ##EQU1##
where I is the transmitted light power, I.sub.0 is the light power incident on the flow cell, .alpha. is the molar absorptivity of the sample, B is the path length of the light in the flow cell (in centimeters), and C is the sample concentration (in moles per liter). The detector output is usually in terms of Absorbance (A) which is defined as the product .alpha. B C and is proportional to both the sample concentration, C, and the path length, B. The longer the path length, the larger the detector output signal for a given sample concentration.
In conventional flow cells, light that strikes the lateral wall of the flow cell is partially lost due to absorption and scattering at the wall. This lost light causes an increase in noise in the output signal of the detector. The lateral dimension or diameter of the flow cell can be increased to reduce the fraction of light striking the lateral wall, but this increases the volume of the flow cell in proportion to the radius squared. A larger cell volume results in spreading out or dispersion of a sample peak and loss in chromatographic resolution in HPLC and a similar loss in resolution in CZE. In practice, this loss in resolution limits conventional flow cells to path lengths of the order of 6 to 10 mm for HPLC and even shorter for CZE because of the narrower sample peaks or smaller peak volumes associated with CZE.
Accordingly, it has long been desired to produce flow cells capable Qf longer path lengths without an excessive increase in light loss or cell volume. This desire may be realized by providing that the interior wall of the flow cell comprises or is covered with a low refractive index polymer so that light striking the coated wall is totally infernally reflected back into the cell bore, and light-piped along the cell bore. The basic requirement for light-piping (i.e., achieving total internal reflectance of light) is that the refractive index of the interior wall be less than that of the liquid in the flow cell. Water has the lowest refractive index (in the UV range of the spectrum for wavelengths between 190 nm and 300 nm) of liquids commonly used in HPLC and CZE, so the refractive index of the inner wall should be less than that of water. A further requirement of the inner wall is that it be reasonably transparent at the wavelengths used in the measurement of light absorption in the flow cell. While light does not propagate in the inner wall when total internally reflected, an evanescent wave is established along the surface that will absorb light power if the wall material is not transparent.
Light-piping in a liquid is not a new concept. Commercial liquid light pipes are available, but these usually contain a high refractive index liquid so that polymer coating of TEFLON.RTM. TFE and TEFLON.RTM. FEP both of which are available from DuPont Polymers of Wilmington, Del., will effectively pipe light. However, these long available polymers will not effectively pipe light in low refractive index liquids like water as their indices of refraction are greater than that of water.
Recently, new fluoropolymers have become available having indices of refraction which are less than that of water. Such fluoropolymers are available from DuPont as TEFLON.RTM. AF. Gilby et al, U.S. Pat. No. 5,184,192, and Liu, U.S. Pat. Nos. 5,416,879 and 5,444,807, all teach flow cells which employ these new fluoropolymers. Liu broadly describes methods of manufacturing such flow cells either by forming the fluoropolymer into rigid tubing or coating the internal walls of a tube with the fluoropolymer. Gilby et al teach alternative methods for forming the flow cells, either by depositing a coating of the fluoropolymer from a solvent or coating the exterior surface of a soluble tube with the fluoropolymer, encapsulating the coated tube in a polymer matrix, and then dissolving the tube.
However, the methods heretofore used in the art have not been entirely successful in producing a flow cell which totally internally reflects light because the large aspect ratio of tube length to tube diameter in combination with the high surface tension of the fluoropolymer makes the coating of the fluoropolymer in the one process and the dissolution of the tube in the other process extremely difficult. Thus, the prior art processes are unable to control either the internal diameter, the surface finish, and the thickness of the material. Accordingly, the need remains for an improved process for the manufacture of a flow cell with walls having an index of refraction lower than that of water and which substantially totally internally reflects light along the cell bore.